The objective of this study was to identify blood metabolites associated with variation primarily in residual feed intake (RFI) in two populations of beef steers at the University of Guelph and University of Alberta, Canada, representing the discovery and validation populations, respectively. Other productivity performance traits including average body weight (ABW), average feed intake (AFI), dry matter intake (DMI) and average daily gain (ADG) were also investigated. In the discovery population, blood plasma samples were obtained from 32 steers (16 high- and 16 low-RFI, from a population of 112 steers and maximizing the divergence between groups for RFI) at three periods 1, 2 and 3, corresponding to weeks 2, 6 and 10 respectively of the 140. d feeding and performance test. In the validation population, blood samples were obtained from 20 (10 high and 10 low RFI) steers from periods 1 and 2 corresponding to weeks 2 and 6 of a 90. d feeding test period. Metabolite concentrations in plasma were determined using nuclear magnetic resonance (NMR) and multiple regression analysis was performed in SAS 9.1. Creatine and glycine were associated (P<0.05) with RFI in period 1 accounting for 36.3% of the phenotypic variation in RFI. At period 2, threonine, carnitine, acetate, creatine, phenylalanine, lysine, citrate, betaine, glutamate and hippurate were significant (P<0.05) and accounted for 74.2% of the variation in RFI. At period 3, hydroxyisobutyrate, tyrosine and formate were significant (P<0.05) and accounted for 52.1% of the variation in RFI. In the validation population, three metabolites (creatine, carnitine and hippurate) were significant (P<0.05) in both discovery and validation populations and these three metabolites accounted for 32% of the phenotypic variation in RFI in the validation population. Some of the metabolites associated with RFI were also associated with other performance traits discussed in subsequent sections. Metabolic networks for RFI in each period were reconstructed using IPA and suggested that the biological processes associated with RFI were involved in energy and protein metabolism as well as metabolism of urea and methane. The analysis of metabolites and evaluation of biological processes create a better understanding of the metabolic processes that affect RFI. Upon further validation, these indicators may have potential to be utilized as biomarkers to enhance the selection of beef cattle.